Purpose:
Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation, a process that requires retro-translocation and ubiquitination mediated by a lumenal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER-stress induced cell death. We sought to evaluate whether distinct myocilin mutants were triaged similarly by the ER chaperones Grp94 and Grp78.

Methods:
We used a cell culture model of mutant myocilin accumulation to evaluate the effects of chaperones on mutant myocilin triage. We used siRNA, over-expression, Western blot, immunofluorescence, sub-cellular fractionation and drug treatments for these studies.

Results:
Using targeted knockdown strategies, we determined that Grp94, the ER equivalent of Hsp90, specifically recognizes mutant myocilin, triaging it through ER-associated protein degradation (ERAD). However, not all mutant myocilin species are handled the same way. The insolubility of mutant myocilin species that cause a more aggressive form of primary open angle glaucoma was enhanced by Grp94 depletion. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that beta-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD towards a more robust clearance pathway or towards further aggregation, suggesting that the kinetics of aggregation and possibly the structure of distinct myocilin mutants are important for successful handling of amyloidogenic proteins in ER.

Conclusions:
Grp94 is an essential player in deciding the fates of mutant myocilin species. The balance of Grp94 levels may be an important factor in determining whether distinct mutant myocilin species are cleared from the cell or allowed to aggregate. Regardless, it is possible that both of these outcomes are ultimately protective for the cell. Therefore therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.